Taxonomic Applicability

Sex Applicability

Life Stage Applicability

Key Event Relationship Description

NMDARs can be activated indirectly through initial activation of KA/AMPARs as it happens in the case of DomA exposure. DomA is an agonist of
presynaptic and postsynaptic KARs and sustained activation of these receptors by DomA results in massive ion flux and excessive release of glutamate
from excitatory terminals causing depolarization of the postsynaptic neuron (as descibed in MIE). Upon this depolarization the Mg2+ block is removed
from the pore of NMDARs, resulting in their activation allowing sodium, potassium, and, importantly, calcium ions to enter into a cell. The sustained
exposure to DomA causes pathological overactivation of NMDARs.
In the case of exposure to glufosinate NMDARs activation is triggered by direct, sustained binding of glufosinate to the NMDARs.

Evidence Supporting this KER

Biological Plausibility

NMDARs are unique among ligand-gated ion channels in that their activation requires binding of two co-agonists, glycine and endogenous
neurotransmitter, L-glutamate. Physiologically, however, glycine and glutamate have distinct functions. While L-glutamate is released from specific
presynaptic terminals, low concentrations of ambient glycine present at the synapse are thought to be sufficient to allow receptor activation.
There is a clear understanding that binding of glutamate or its analogue will activate NMDA receptor (accepted dogma). The prolonged activation of
NMDARs will lead to a pathological over-activation of a receptor leading to excitotoxicity (minor role of KA/AMPARs), allowing high levels of calcium
ions to enter the cell. However, KA/AMPARs play an important role for indirect NMDAR activation since (almost always) an initial activation of these
receptors triggers depolarization of postsynaptic neurons that relieves the block of the channel pore by Mg2+, resulting in NMDAR activation.
NMDA receptors are formed by a ligand binding domain (LBD) and an ion channel that are considered the core structural and functional elements of
the receptors. There is a clear understanding of how agonist binding leads to channel opening that relies on structural (e.g. crystallography or NMR)
and functional (e.g. UV and IR spectrometric measurements) experimental studies of the water-soluble LBD combined with functional studies of the
intact receptor. After the initial agonist binding, a conformational change—so-called clam shell closure—that prevents agonist dissociation occurs
followed by a conformational change in the ion channel that is tightly coupled to that in the LBD (reviewed in Traynelis et al., 2010).
Consequently it can be stated that there is a clear structural and functional mechanistic understanding in this KER between MIE (Binding of agonist to
glutamate ionotropic receptors) and KE1, NMDAR overactivation that, as explained above, can be triggered by direct binding to NMDAR or indirectly,
through initial activation of KA/AMPARs as it happens in the case of exposure to glufosinate and DomA respectively, two stressors described in this
AOP.

Indeed, domoic acid has a very strong affinity for the ionotropic glutamate receptors, the activation of which results in excitotoxicity, initiated by an
integrative action of ionotropic receptors at both sides of the synapse blocking the channel from rapid desensitization. It has a synergistic effect with
endogenous glutamate and it acts mainly as an agonist for presynaptic and postsynaptic kainate receptors. Activation of ionotropic receptors leads to
the influx of Na+, K+ and Ca2+, particulary after activation of NMDARs. In combination with the inhibitory GABA neurotransmitter, glutamate
contributes to the control of overall neuronal excitability.

Gufosinate (GLF) triggers alterations in glutamatergic signaling through direct binding and activation of NMDARs (Lantz et al., 2014: Matsumura et al.,
2001). GLF agonist action at the NMDAR is expected to occur through interaction with the glutamate binding site and requires binding of the glycine
co-agonist as well as release of the magnesium block from the channel pore. Additionally, the possible inhibition by GLF of the high affinity glutamate
re-uptake transporter, especially GLT-I was studied to determine whether GLF could increase the levels of endogenous glutamate at the synaptic cleft,
resulting in over activation of NMDARs. Such mechanism was excluded by Lantz (Lantz et al., 2014) but suggested by other studies (Watanabe and
Sano, 1998).

Empirical Evidence

There is well established understanding of NMDAR activation by endogenous agonist glutamate that happens in the absence of the Mg2+ block under
conditions of depolarized post-synaptic membrane(accepted dogma) (Blanke et al., 2009a and b; Enoki R, et al.,2004).

Single channel behavior of NMDARs from hippocampal CA1 neurons was studied using very low glutamate concentrations to improve temporal
resolution of individual glutamate binding events. Openings resulting from individual receptor activations showed surprising complexity: they consist of
a long cluster of bursts of openings. Furthermore, the NMDARs appeared to have different gating modes, occasionally entering periods of very high
open probability (Gibb et al., 1991).
Single channel analysis also provided insight in how NMDARs function at the synapse. In response to a brief pulse of glutamate, mimicking synaptic
release, NMDARs activate slowly over hundreds of milliseconds and continue activating long after all glutamate has been removed from the synaptic
cleft, thereby briefly “memorizing” the occurrence of a synaptic input. Single channel analysis of NR1 and NR2A receptors indicates that after a brief
pulse of glutamate, receptors enter a high affinity closed state from which either channel opening or agonist unbinding occurs with approximately
equal probability (Popescu et al., 2004). A single synaptic event is therefore expected to only partially activate NMDARs. Consequently, a closely
spaced second pulse of agonist is able to further increase the open probability, endowing the NMDAR with an ability to decode synaptic input
frequency.

Domoic acid is an agonist for presynaptic and postsynaptic kainate receptors, however indirectly also activates NMDA receptors. Kainate receptors
are localized both at presynaptic and postsynaptic sites. At presynaptic sites, they directly affect transmitter release from both excitatory and inhibitory
neuron terminals. At postsynaptic sites, kainate receptors lead to cell depolarization, which would bring the neuron closer to its spike firing threshold.
By having this dual localization, kainate receptors help in the control of neuronal excitability. However, sustained activation of postsynaptic kainate
receptors by domoic acid results in massive ion flux and excessive release of glutamate from excitatory terminals. The released glutamate in turn
activates NMDA receptors, which have lost their physiologic Mg2++ block because of domoic acid–induced depolarization. The final event is an
increase of NMDA-mediated Ca2++ flux and subsequent activation of intracellular pro-oxidative cascades and ion imbalances, eventually leading to
excitotoxicity-mediated neuronal death (Babot et al., 2005;Giordano et al., 2006).

Kainate receptors are widely expressed in the hippocampus. Glutamatergic granule cells in the hippocampus express these receptors, suggesting that
cell death found after domoic acid intoxication may be produced by hyperstimulation of NMDA receptor after glutamate is released in excess. In
agreement with this hypothesis, the seriously damaged CA3 area of the hippocampus receives projections from hippocampal granule cells. Qiu and
Curras-Collazo (Qiu et al., 2006a) elegantly demonstrated that domoic acid first targets kainate receptors in the hippocampus by blocking its effects in
vivo with a kainate receptor antagonist. The sequential involvement of distinct glutamate receptors was confirmed and further elucidated in rat mixed
cortical cell and hippocampal slice cultures (Jakobsen et al., 2002; Qiu et al., 2006b).

Using primary cultures of rodent cerebellar granule cells, an in vitro model mainly constituted by glutamatergic neurons that express both NMDA and
kainate receptors it was proved that domoic acid increased glutamate release, intracellular calcium, and cell death, which were prevented by kainate
and NMDA receptor antagonists (Berman and Murray, 1997; Vale-Gonzalez et al., 2006) confirming that DomA toxicity is mediated by both KA and
NMDARs.

Glufosinate (GLF) and its primary metabolite N-acetylglufosinate (NAcGLF) interaction with NMDARs was studied in the primary culture of rat cortical
neurons by performing [3H]CGP 39653 binding experiments. The results showed that their binding affinity to NMDAR (IC50, GLF 668 uM and NAcGLF
approximately 100 uM) corresponded to the concentration that produce the highest increase of mean firing rate. Furthermore, they produced biphasic
MFR profile, specific to NMDAR agonists. The obtained results suggest that GLF and NAcGLF can produce both effects,excitatory and inhibitory on
network activity through direct activation of NMDARs (Lantz et al., 2014).

Direct activation of NMDARs by GLF is also suggested by in vivo studies where three NMDA receptor antagonists, dizocilpine, LY235959, and Compound
40, and AMPA/KA antagonist, NBQX, were co-administrated with glufosinate ammonium (80 mg/kg, intraperitoneally) in mice. Statistical analyses
showed that the NMDA receptor antagonists markedly inhibited the GLF-induced convulsions, while the AMPA/KA receptor antagonist had no effect.
These results suggest that the convulsion caused by glufosinate ammonium were mediated through activation of NMDA receptors (Matsumura et al.,
2001).

Uncertainties and Inconsistencies

The increase in MFR induced by GLF in neuronal networks was significantly blocked by MK-801 but not entirely suggesting that GLF can increase
activity in the MEA system through non-synaptic NMDARs, since these are not blocked by MK-801.
It is not entirely clear whether GLF can work through an inhibition of the glutamate reuptake transporter, GLT-I, increasing the concentration of
endogenous glutamate at the synaptic cleft and subsequently resulting in over activation of NMDARs (Lantz et al., 2014: Watanabe and Sano, 1998).
Further studies are necessary to determine whether this alternative mechanism of GLF-induced NMDAR overactivation takes place.
Additionally GLF also modulates glutamine synthetase (GS) activity. Since, astrocytic GS in the brain participates in the metabolic regulation of glutamate (endogenous agonist of NMDAR) it is not clear if this pathway contributes to NMDAR activation too.

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second?
Are there known modulators of the response-response relationships?
Are there models or extrapolation approaches that help describe those relationships?

To predict how potent an agonist can be, it is usually based on the half maximal effective concentration (EC50) that induces the currents through
NMDA receptors of brain slices and cells (or in recombinantly expressed proteins of these receptors). Traynelis et al. 2010 summarised the IC50 values
for agonists of the different NMDA receptor subunits.
The activation effect (efficacy) of agonist on NMDA receptor have been found to be dependent on:
-the type of subunits that form the NMDA receptor
-the chemical structure of the agonist
-the binding site of a receptor that the agonist prefers
-how tightly an agonist binds to the receptor (affinity)
Glufosinate and its primary metabolite N-acetylglufosinate NAcGLF bind to the NMDAR with the following affinity: the IC50 value for GLF was 668 mM
and for NAcGLF was about 100 mM.

Response-response Relationship

Time-scale

Known modulating factors

Known Feedforward/Feedback loops influencing this KER

Domain of Applicability

Various studies suggest the existence of functional NMDA-like receptors in invertebrates (Xia et al., 2005). Fly and rodent NMDARs exhibit several
important differences (Murphy and Glanzman, 1997). The expression and function of NMDA receptors in rodent and primates is well characterized in
the existing literature.